Scanning mutagenesis of the voltage-gated sodium channel NaV1.2 using base editing

Juan Lorenzo B. Pablo*, Savannah L. Cornett, Lei A. Wang, Sooyeon Jo, Tobias Brünger, Nikita Budnik, Mudra Hegde, Jean Marc DeKeyser, Christopher H. Thompson, John G. Doench, Dennis Lal, Alfred L. George, Jen Q. Pan*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

It is challenging to apply traditional mutational scanning to voltage-gated sodium channels (NaVs) and functionally annotate the large number of coding variants in these genes. Using a cytosine base editor and a pooled viability assay, we screen a library of 368 guide RNAs (gRNAs) tiling NaV1.2 to identify more than 100 gRNAs that change NaV1.2 function. We sequence base edits made by a subset of these gRNAs to confirm specific variants that drive changes in channel function. Electrophysiological characterization of these channel variants validates the screen results and provides functional mechanisms of channel perturbation. Most of the changes caused by these gRNAs are classifiable as loss of function along with two missense mutations that lead to gain of function in NaV1.2 channels. This two-tiered strategy to functionally characterize ion channel protein variants at scale identifies a large set of loss-of-function mutations in NaV1.2.

Original languageEnglish (US)
Article number112563
JournalCell reports
Volume42
Issue number6
DOIs
StatePublished - Jun 27 2023

Keywords

  • CP: Neuroscience
  • CRISPR
  • NaV1.2
  • base editing
  • channelopathy
  • electrophysiology
  • ion channels
  • mutational screening
  • neurodevelopmental disorder
  • variants to function
  • voltage-gated sodium channel

ASJC Scopus subject areas

  • General Biochemistry, Genetics and Molecular Biology

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